This invention relates generally to connective tissue growth factor (CTGF) and to receptors thereof.
Growth factors are a class of secreted polypeptides that stimulate target cells to proliferate, differentiate, and organize developing tissues. Typically, a growth factor""s activity is dependent on its ability to bind to specific receptors, thereby stimulating a signaling event within the cell. Examples of some well-studied growth factors include platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor beta (TGF-xcex2), transforming growth factor alpha (TGF-xcex1), epidermal growth factor (EGF), and fibroblast growth factor (FGF). Efforts to characterize the receptors corresponding to these growth factors are ongoing and have met with varying degrees of success.
Low Density Lipoprotein Receptor-Related Protein (LRP)
A low density lipoprotein receptor-related protein (LRP) has been reported previously in the literature. (See, e.g., Kristensen et al., 1990, FEBS Lett. 276:151-155.) This protein is also known as the xcex12-macroglobulin receptor. (See, e.g., Strickland, D. K. et al. (1990) J. Biol. Chem. 265:17401-17404; and Herz et al. (1988) EMBO J. 7:4119-4127.) LRP is encoded by a protein sequence of about 4544 amino acids in length (human mRNA for LDL-receptor related protein, GenBank Accession No. X13916; human LDL-receptor related precursor protein, GenBank Accession No. CAA32112; Herz et al., id; Myklebost et al. (1989) Genomics, 5:65-69, each of which references is incorporated by reference herein in its entirety.) The mature protein consists of a 4419 amino acid ectodomain, a twenty-five amino acid transmembrane domain and a 100 amino acid intracellular domain. As reported in the literature, the ectodomain contains a furin cleavage site which is processed during transport from a late Golgi compartment, producing a 510 kDa xcex1 subunit that is noncovalently associated with an 85 kDa xcex2 subunit anchored to the membrane through the transmembrane sequence. (See, e.g., Strickland, D. K. et al., supra.)
The xcex12-macroglobulin receptor has been widely studied and a number of subdomains have been identified within the xcex1 and xcex2 subunits. These subdomains include twenty-two EGF-like domains, of which two such domains are Ca2+ binding, eight are EGF precursor spacer regions, and thirty-one are LDL receptor ligand-binding repeats. Additionally, thirty-one copies of complement-type repeats arranged in four clusters spanning the receptor sequence have been identified. It has been reported that the receptor protein sequence is highly conserved (more than 97% homology) between the human and murine systems. (See, e.g., Van Leuven et al. (1993) Biochim. Biophys. Acta., 1173:71-74.)
The cytoplasmic domain of the xcex12-macroglobulin receptor has no homology with known protein kinase domains. Genetic analysis of the protein function by disruption of the xcex12-macroglobulin receptor gene in order to create xcex12-macroglobulin receptor-deficient mice has indicated that the protein is essential during development. A number of ligands have been reported to bind to the xcex12-macroglobulin receptor, including xcex12 macroglobulin, activated; apolipoprotein E (apo E); low density lipoprotein, apo E enriched; Pseudomonas exotoxin A; receptor-associated protein (RAP); plasminogen activator inhibitor (PAI) I; thrombin-PAI complex; tissue plasminogen activator (tPA); urokinase plasminogen activator (uPA); thrombospondin I; lipoprotein lipase; hepatic lipase; lactoferrin; pregnancy zone protein; xcex11-inhibitor-3; xcex11-inhibitor-3/xcex11 microglobulin complex; xcex2 amyeloid precursor protein; suramin; and vitellogenin. The adaptor molecule mDab1 has been shown to bind to the cytoplasmic tail of LRP in neuronal cells. (See, e.g., Le, N., and M. A. Simon (1998) Mol. Cell. Bio. 18:4844-54; and Trommsdorff, M. et al. (1998) J. Biol. Chem. 273:33556-60.) When tyrosine-phosphorylated, mDab1 binds non-receptor tyrosine kinases, such as src, fyn, and abl. (See, e.g., Howell, B. W. et al. (1997) Embo Journal. 16:121-32.) Another member of this family, Dab2, is expressed more widely, and has recently been shown to bind Grb2, an adaptor protein which couples tyrosine kinase receptors to Sos which is part of Ras signaling cascade. (See, e.,g., Xu, X.X. et al. (1998) Oncogene. 16:1561-9; and Fazili, Z. et al. (1999) Oncogene. 18:3104-13.) Recent findings indicate broad physiological functions for LRP and other members of the LDL receptor family, suggesting that interfering with any associated signaling cascade would provide methods of modulating activities associated with LRP. (Gotthardt et al. (2000) J. Biol. Chem., 275:25616-25624.)
Connective Tissue Growth Factor (CTGF)
Connective tissue growth factor (CTGF) has been reported and described previously. (See, e.g., U.S. Pat. No. 5,408,040; Bradham et al., 1991, J. Cell Biology 114:1285-1294.) CTGF is characterized as a polypeptide that exists as a monomer with a molecular weight of approximately 36 to 38 kD. CTGF has been shown to be one of seven cysteine-rich secreted proteins belonging to the CCN family, which includes CTGF, cyr-61, and nov. (See, e.g., Oemar et al. (1997) Arteriosclerosis, Thrombosis and Vascular Biology 17(8):1483-1489.) CTGF is the product of an immediate early response gene that codes for a protein consisting of four modules and one signal peptide. (See, e.g., Oemar et al. (1997), supra.) The four modules include an insulin-like growth factor (IGF) binding domain, a von Willebrand factor type C repeat most likely involved in oligomerization, a thrombospondin type 1 repeat believed to be involved in binding to the ECM, and a C-terminal module which may be involved in receptor binding. Recent reports suggest that certain fragments of the whole CTGF protein possess CTGF activity. (See, e.g., Brigstock et al. (1997) J. Bio. Chem. 272(32):20275-282; International Publication No. WO 00/047114; and International Publication No. WO 00/047130, each of which references is incorporated herein by reference in its entirety.) Human, mouse, and rat CTGF are highly conserved, with greater than 90% amino acid homology (Bork (1993) FEBS Lett. 327:125-130), and a molecular weight of about 38 kDa (Bradham et al. (1991) J Cell Biol. 114:1285-1294). It was recently shown that the promoter of the CTGF gene contains a novel TGF-xcex2 responsive element. (Grotendorst et al. (1996) Cell Growth and Differentiation 7:469-480.)
CTGF plays a role in the production of collagen and other extracellular matrix proteins. CTGF has mitogenic and chemotactic activity, and its effects have been observed in connective tissue cells, e.g., fibroblasts, as well as in a number of other cell types. The ability of CTGF to effect cell proliferation and motility have led to its implication in a variety of disorders associated with excess growth and increased deposition of extracellular matrix, including disorders such as fibrosis, cancer, angiogenesis, and other proliferative disorders. For example, CTGF appears to be a causal factor in skin fibrosis and in atherosclerosis. (See, e.g., Igarashi et al. (1995) The Journal of Investigative Dermatology 105:280-284; Igarashi et al. (1996) The Journal of Investigative Dermatology 106:729-733; Oemar et al. (1997) Circulation 95:831-839).
CTGF is therefore an attractive target for the development of therapeutic agents useful in the treatment of a number of connective tissue diseases and proliferative disorders. The desirability of modulating, and, preferably, inhibiting, CTGF activity as a method for treating fibrotic diseases and disorders has been previously described. (See, e.g., PCT Application No. PCT/US96/08140.) Various inhibitors of CTGF activity, including peptides, antibodies to CTGF, and the like, have been described and are reported to have potential therapeutic effectiveness in the treatment of fibrotic disease. (Id.) It has also been determined that CTGF is capable of inducing bone and cartilage growth and tissue repair, such as wound healing. (See, e.g., U.S. Pat. No. 5,408,040 and PCT Application No. PCT/US96/08210.) Biological activities attributed to CTGF include stimulating fibroblast proliferation (Kothapalli et al. (1 997) Cell Growth Differ. 8:61-68; Frazier et al. (1996) J. Invest. Dermatol. 107:404-411; Kothapalli et al. (1998) FASEB J. 12:1151-1161; Kothapalli and Grotendorst (2000) J. Cell. Physiol. 182:119-126), cell adhesion, migration, angiogenesis (Babic et al. (1999) Mol. Cell. Biol. 19:2958-2966; Shimo et al. (1999) J. Biochemistry (Tokyo) 126:137-145), stimulating the expression of extracellular matrix components, such as collagen, fibronectin, and 5-integrin (Frazier et al. (1996) J. Invest. Dermatol. 107:404-411), and in some cells, apotosis (Hishikawa et al. (1999) Eur. J. Pharmacol. 385:287-290; Hishikawa et al. (1999) J. Biol. Chem. 274:37461-37466). High expression of CTGF has also been associated with wound healing and granulation tissue formation (Frazier et al. (1996) J. Invest. Dermatol. 107:404-411; Moir et al. (1999) J. Cell. Physiol. 181:153-159). In embryonic development, CTGF has been observed specifically at sites of endochondral ossification (Nakonnishi and Takigawa (1999) Seikagaku 74:429-432), and embryo implantation within the uterus (Surveyor et al. (1998) Biol. Reprod. 59:1207-1213; Surveyor and Brigstock (1999) Growth Factors 17:115-124). CTGF mRNA and protein over-expression have been localized to affected tissues in disease states, including scleroderma and keloid fibroblasts (Igarishi et al. (1996) J. Invest. Dermatol. 106:729-733), mesangial cells within renal fibrosis (Riser et al. (2000) J. Am. Soc. Nephrol. 11:25-38), pancreatitis (di Mola et al. (1999) Ann. Surg. 230:63-71), bleomycin-induced pulmonary fibrosis (Lasky et al. (1998) Am. J. Physiol. 275:L365-371), systemic sclerosis (Igarashi et al. (1995) Invest. Dermatol. 105:280-284; Sato et al. (2000) J. Rheumatol. 27:149-154), fibrous stroma of mammary tumors (Frazier and Grotendorst (1997) Int. J. Biochem. Cell. Biol., 29:153-161), advanced atherosclerotic lesions (Oemar et al. (1997) Circulation 95:831-839), the infarct zone of myocardial infarction (Ohnishi et al. (1998) J. Mol. Cell. Cardiol. 30:2411-2422), inflammatory bowel disease (Darnmeier et al. (1998) Int. J. Biochem. Cell. Biol. 30:909-922), and desmoplastic malignant melanoma (Kubo et al. (1998) Br. J. Dermatol, 139:192-197. The over-expression of CTGF in tissue has been highly correlated with the onset and extent of renal and liver fibrosis (Ito et al. (1998) Kidney Int. 53:853-861; Paradis et al. (1999) Hepatology 30:968-976. Therefore, understanding the role of CTGF within these disease states, therefore, is of great importance.
Substantial efforts have been directed to the isolation, characterization, and use of CTGF as a target in treating a variety of disorders. The potential benefits of the ability to modulate CTGF expression and activity, either to inhibit the overproduction of connective tissue and extracellular matrix, such as when treating fibrotic and other proliferative disorders, or to induce bone, tissue, and cartilage repair, when increased CTGF expression and activity would be desired, are evident. However, despite ongoing efforts, there has been no report of the identification, characterization, or isolation of receptors to CTGF. Likewise, means of modulating the activity of such receptors in order to achieve specific therapeutic effects, such as by administration of antibodies or other agents capable of effecting CTGF receptor activity, have not been reported in the literature.
In summary, CTGF plays a significant role in the normal development, growth, and repair of human tissue. The ability to enhance, inhibit, or otherwise modulate the activity or expression of CTGF could therefore be a valuable therapeutic tool. Affecting the ability of CTGF to bind to its receptor could be a useful means of modulating CTGF activity or expression. Therefore, there is a need for identification of a CTGF receptor and for means of modulating CTGF receptor activity.
The present invention is based on the discovery that CTGF binds to a particular protein, the low density lipoprotein receptor-related protein (LRP), also known as the xcex12-macroglobulin receptor, and the identification of LRP as a CTGF receptor. There has been no previous report that CTGF or fragments thereof bind to LRP. This discovery satisfies a need in the art as the receptor may be used to modulate and to identify other agents that modulate CTGF activity, and can provide a basis for the development of new therapeutic tools and methods for treatment of CTGF-associated disorders.
The present invention relates to the identification of a CTGF receptor and to methods of diagnosis, treatment, and screening.
In one aspect, the present invention provides a method of treating or preventing a CTGF-associated disorder, the method comprising administering to a subject in need an effective amount of an agent that effects the expression or activity of a CTGF receptor or fragments or subunits thereof. In a further embodiment, the method of treatment or prevention comprises administering to a subject in need an effective amount of an agent that inhibits the activity or expression of a CTGF receptor or fragments or subunits thereof. The agent can be, for example, an antibody that specifically binds to a CTGF receptor or fragments or subunits thereof, an antisense oligonucleotide having a sequence that binds to a sequence encoding a CTGF receptor or fragments or subunits thereof, or a small molecule. The present methods can be directed to the treatment of various disorders, including, for example, proliferative disorders, fibrotic disorders, sclerotic disorders, cancer, and angiogenesis.
In some methods according to the present invention, it can be desirable to increase the expression and activity of CTGF. Therefore, in one aspect, the present invention provides a method of treating or preventing a CTGF-associated disorder associated with decreased expression or activity of CTGF, the method comprising administering to a subject in need an effective amount of an agent that increases the activity or expression of a CTGF receptor or fragments or subunits thereof. In one embodiment, the agent is a CTGF receptor or fragments or subunits thereof.
The present invention further provides a method for identifying an agent that modulates the expression or activity of a CTGF receptor, the method comprising contacting a candidate compound with the CTGF receptor; detecting the level of CTGF receptor expression or activity in the sample; and comparing the level of CTGF receptor expression or activity in the sample to a standard level of CTGF receptor expression or activity.
In another aspect, the present invention encompasses pharmaceutical compositions comprising an effective amount of an agent that modulates the expression or activity of a CTGF receptor or fragments or subunits thereof and a suitable carrier. The agent can be a CTGF receptor agonist or antagonist, for example, or can comprise a CTGF receptor or fragments or subunits thereof and a suitable carrier.
In a further embodiment, the invention provides a method for diagnosing a CTGF-associated disorder, or identifying a predisposition or susceptibility to such a disorder, in a subject, the method comprising obtaining a sample from the subject; detecting the level of CTGF receptor expression or activity in the sample; and comparing the level of CTGF receptor expression or activity in the sample to a standard level of CTGF receptor expression or activity. In a preferred embodiment, the sample from the subject is a urine sample. In one aspect, the present invention provides for a diagnostic kit for use in diagnosing a CTGF-associated disorder, or identifying a predisposition or susceptibility to such a disorder, the kit comprising a means for detecting the level of CTGF receptor expression or activity in a sample; and a means for measuring the level of CTGF receptor expression or activity in the sample. In a preferred embodiment, the diagnostic kit of claim 17, wherein the sample is a urine sample.
Various other embodiments of the present invention are described herein.